STK4 protein expression pattern follows different trends in endometrioid and serous endometrial adenocarcinoma upon tumor progression

In a previous study, we showed that serine/threonine-protein kinase 4 (STK4) is involved in the control on proliferation and migration of endometrial cancer (EC) cells in vitro. In the present paper, we studied STK4 expression in EC tissues from a large cohort of patients to determine whether STK4 can serve as a marker for the aggressiveness and prognosis of EC. Tissue samples from patients with EC were examined for tumor type, grade, and stage. The STK4 protein expression in EC cells was assessed by immunohistochemistry and related to clinicopathological data of patients, such as progression and patient survival rate. The STK4 mRNA levels and its relation to the survival rate were analyzed also in publicly available databases. The STK4 gene expression was low at both, the mRNA and protein levels in EC, especially in serous tumors. Comparison of STK4 expression with the patient survival rate shows that the higher expression is associated with worse prognosis in serous EC, while no such dependence was found in endometrioid EC. Hence, the determination of the SKT4 expression pattern could be used as a putative prognostic marker for serous EC.

www.nature.com/scientificreports/ expression of the programmed death-ligand 1 (PD-L1) significantly correlates with cancer progression 6 . Obviously, the search for new markers is still an important and urgent task. Earlier, our research group showed that STK4 (NP_006273), also designated as a mammalian sterile 20-like 1 (MST1) protein, is involved in the control on proliferation and migration of EC cells, using a proteomics approach 4,7,8 . STK4 shows structural homology with the budding yeast sterile 20 protein (Ste20). It was demonstrated that knocking down the YHL007C gene in the yeast genome resulted in the inability of a cell to arrest cell cycle 9 .
Importantly, we found that overexpression of the STK4 protein in EC cell lines enhanced migration capability 8 . Increased capacity for migration can be associated with the fact that STK4 is involved in the control on cell polarity, i.e., it can promote epithelial to mesenchymal cell transition (EMT), which is essential for migration and invasiveness of cancerous cells.
As we mentioned above, the CTNNB1 gene, encoding β-catenin, is often mutated in endometrioid EC 3 . β-catenin is involved in the EMT control and plays a vital role in maintaining tissue architecture and regulating E-cadherin expression [10][11][12] . It was demonstrated that patients with E-cadherin-negative endometrioid EC had a worse prognosis 13,14 . Earlier, we showed a low expression of E-cadherin, together with diminished pan-keratin and β-catenin, in EC cell lines, especially when the MRPS18-2 protein was overexpressed 15 .
Because STK4 overexpression leads to an enhanced migration activity of EC cells in vitro, which is important for tumor progression, we wanted to study the STK4 expression pattern in EC tissues from a large cohort of patients. In the present paper we aimed to answer the question whether STK4 can serve as a marker for the aggressiveness and prognosis of EC.

Results
Patients and tumor characteristics. The age range of the cohort was 36-92 years. A significant age increase was observed for patients with more advanced EC; this was valid when all patients were grouped by tumor grade (Supplementary Fig. S1A). Of note, patients with serous cancers were older than patients with endometrioid EC (78.5 ± 2.5 and 70.4 ± 6.9, respectively, Supplementary Fig. S1C). ER expression tended to decrease with tumor progression, whereas PR expression and body mass index (BMI) were quite similar in all patient groups ( Supplementary Fig. S1D,E). STK4 protein expression in endometrial cancer. The STK4 protein signal was localized mainly in the nuclei of EC cells. A small proportion of STK4 was also found in the cytoplasm (Fig. 1).
When the intensity of the STK4 protein signal was compared among EC samples, the significantly lower expression levels were observed in the Type 2 tumors, compared with the Type 1 ECs (Fig. 2A). In endometrioid ECs (all samples, that belongs to the Types 1 and 2) the STK4 expression levels were diminished upon tumor progression, i.e., with an increase of the tumor grade (Fig. 2B). This phenomenon was even more apparent when samples of serous EC were included in statistical calculations (Fig. 2C). The STK4 levels were quite similar across different stages of the Type 1 EC samples (Fig. 2D). When all samples (Type 1 and Type 2) were included in calculations, the trend for the lowering of the STK4 signal levels with the more advanced stage was observed (Fig. 2E).
Next, we wanted to analyze an expression pattern of the STK4 gene at mRNA levels in the publicly open databases. To do so, an analysis of the available data at the Oncomine portal was performed. The STK4 gene expression was relatively low at mRNA levels. Moreover, no significant gene copy number changes were detected in clinical tumor tissue samples of EC patients, compared with normal endometrium (Fig. 3A-D).
Next, we asked a question whether the STK4 gene showed any mutations or alterations in ECs. Importantly, the STK4 gene is very seldom mutated in ECs (in 10 out of 709 tissue samples, i.e., in 1.41% of all studied tumors), as showed an analysis of the mutational status, performed, using the COSMIC database. No loss of heterozygosity was detected either; the gain in the gene copy number was insignificant (in 10 out of 598 tumor samples, i.e., in 1.67% of all studied tumors).
Next, we wanted to compare the present results with the data obtained by us earlier, when the studies on EC cell lines were performed 7,8 . To widen up the range and enlarge the number of cell lines, an analysis on the STK4 gene expression was performed in other cell lines, that were derived from solid tumors of a human reproductive system. To do so, the publicly open data deposited to the GTEX Portal and the Cancer cell line encyclopedia (CCLE) were analyzed. Moreover, these data were related to STK4 expression levels in cell lines generated from cancerous cells of hematological malignancies. The latter are characterized usually by quite fast proliferation.
As was mentioned above, STK4 plays an essential role in the control on cell proliferation. Usually, lymphoid and leukemic cells show a shorter doubling time than cells of solid tumors and most normal cells 16 . Of note, STK4 expression at mRNA levels was lower in cell lines from tumors of human reproductive organs, compared with transformed cells or hematological malignances (Fig. 4A,B).
Intriguingly, the copy number of the STK4 gene was slightly higher in solid tumors than in leukemia and lymphoma cells (Fig. 4C). Hence, it is reasonable to propose that diminished STK4 expression levels despite the higher copy number might be explained by methylation of a promoter region of the STK4 gene. A bioinformatic analysis showed that in Burkitt lymphoma, acute T-cell lymphocytic and myeloid leukemia cell lines the STK4 gene promoter was not methylated, in contrast to tumors of the human reproductive system (Fig. 4D). This probably explains the fact that STK4 expression was much higher in hematological malignancies than in solid tumors. STK4 expression and a patient survival rate. Next, using our data on the STK protein expression pattern, we explored whether STK4 levels are related with the prognosis of a course of the disease. To answer this question, STK4 expression levels were related to the survival rate of individuals bearing EC. As we mentioned already, data on STK4 expression was obtained from IHC staining (Fig. 1 www.nature.com/scientificreports/ in the STK4 protein expression were found among the living and deceased patients with the Type 1 EC (Fig. 5A).
On the contrary, a significantly lower STK4 expression was observed in the deceased individuals with the Type 2 EC, suggesting a positive correlation between high STK4 levels and survival of patients with the Type 2 tumors (Fig. 5B).
Taking into consideration, that to Type 2 tumors belong serous tumors and Grade 3 endometrioid tumors, we separated these samples and analyzed patient survival for 95 samples of endometrioid and 13 samples of serous tumors. First, the median expression was calculated (3.6 relative units (r.u.) for endometrioid and 3.2 r.u. for serous cancers. Then expression values were sorted as high (above median) and low (below median). The opposite trends were observed for endometrioid and serous tumors, concerning relation between the intensity of the STK4 protein signal and survival status ( Fig. 5C; Table 1). Thus, in endometrioid tumors high STK4 expression was associated with higher survival (90% of patients with STK4 expression above median was living), compared with low expression (78% of patients was living). In contrast, only 25% of patients bearing serous cancers were alive in a group with high (above median) STK4 expression ( Fig. 5C; Table 1). Of course, the number of serous cancers was not high (only 13), but we may speculate that the high STK4 expression in serous tumors could be a marker for worse prognosis.

Discussion
STK4 is a human ortholog of the Hippo (Hpo) serine/threonine kinase of D.melanogaster. Mutations in the drosophila gene lead to uncontrolled cell proliferation, and consequently, tissues and organs. The mutated gene was named Hippo, from a "hippopotamus"-like phenotype based on this phenomenon. Later, it was shown that the Hippo pathway is highly conserved in all eukaryotes and that it is crucial to the control of organ size during development, regulating cell proliferation and apoptosis.
The HIPPO pathway is deregulated in many human cancers (reviewed in 17 ). For example, it was shown that one of the oncogenes involved in the HIPPO pathway, YAP1 (NM_001130145), is overexpressed in many solid tumors 18 , such as colon, breast, non-small cell lung 19 , ovarian cancers 20 , and hepatocellular carcinoma 21 . www.nature.com/scientificreports/ Other core members of the HIPPO pathway, such as STK3 and STK, have also been implicated in tumor development. They are considered putative tumor suppressors and can inhibit tumor cell proliferation in colon adenocarcinoma and hepatocellular carcinoma [22][23][24] . The importance of the STK3 and STK4 kinases was demonstrated in animal experiments: deletion of both genes led to embryonic lethality 25 . However, single-gene knockouts showed no defects in pups, in which no tumor formation was observed for over two years. Surprisingly, cells lacking either STK3 or STK4 showed enhanced apoptosis and proliferated slower 25 . These effects probably depend on the microenvironment; hence, they could be tissue specific. As the STK4 gene is extremely rarely mutated in EC, impairment of STK4 function is not the consequence of a mutation in endometrioid EC.
We observed low STK4 gene expression at both the mRNA and protein levels in EC, even more decreased in advanced tumors (Fig. 1).
The analysis of data extracted from the publicly available databases showed that the copy number was slightly decreased for the STK4 gene in cancer cell lines produced from tumors of the reproductive organs. However, these values were higher when compared to the normal two copies in such fast-proliferating tumors, as Burkitt lymphoma or acute T-cell lymphocytic leukemia, in which STK4 expression is relatively high. Earlier, the STK4 gene promoter was reported to be often methylated in soft tissue sarcoma 26 . Moreover, treatment with a methylation inhibitor in glioma cell lines was shown to prevent the loss of STK4 expression 27 . The same phenomenon was demonstrated for EC cell lines. Hence, relatively low STK4 expression at the mRNA levels could be explained by promoter methylation.
Could the loss of STK4 expression be a marker of poor prognosis? Comparison of STK4 expression with patient survival shows that high STK4 protein expression correlates with favorable endometrioid EC prognosis (Fig. 5).
The mosaic picture of the relationship between STK4 expression and cell proliferation is preserved even in cell cultures of various origins in vitro. Earlier, working with EC cell lines, we observed increased migration of cancerous cells in vitro upon overexpression of STK4, together with inhibited cell proliferation 8 . In colon cancer cell lines, overexpression of STK4 resulted in increased apoptosis and decreased proliferation and migration 28 . www.nature.com/scientificreports/ This new panel could be an excellent supplement to PTEN mutations and TP53 accumulation. Apparently, different microenvironment and stimuli influence the cell proliferation and migration rate during EC development. Importantly, serous and endometrioid cancers differ in the underlying molecular mechanisms. Thus, endometrioid cancers show high levels of the wild type TP53 protein. Because most endometrioid EC are ER positive, TP53 is functionally inactivated by ER binding 29,30 . Deregulation of the tissue structure could be due to CTNNB1 mutations and low E-cadherin. As discussed above, overexpression of STK4 led to lower rates of cell proliferation. High levels of STK4 were associated with favorable outcome of endometrioid cancers, in contrast to serous tumors (Figs. 2 and 5). It is known that mutations in the CTNNB1 gene in serous tumors is a very rare event. However, TP53 is mutated in most serous ECs and the latter are hormone-independent, as a rule. Probably, high STK4 levels in cancerous cells of serous tumors plays an opposite role, i.e., overexpression of STK4 leads to faster cell migration and worse patient outcome.
Many proteins, and not identified yet, play an essential role in EC progression. The future approach could be to analyze simultaneously expression of several proteins, implicated in EC development, namely E-cadherin, β-catenin, PKN1, vimentin, and MRPS18-2, together with STK4 and STK3, to predict EC outcome and patient survival better. Importantly, different expression patterns are expected for endometrioid and serous tumors.

Materials and methods
Clinical samples. In the present work, we used a cohort of 108 patients with EC that included tissues samples from 95 patients with endometrioid and 13 with serous EC (see a supplementary Table S1) that were collected at the National Institute of Cancer and RE Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology (IEPOR) of the National Academy of Sciences of Ukraine (NASU) (Kyiv, Ukraine). Included patients had no previous chemotherapy or radiation therapy and had information available on FIGO stage and FIGO grade; as well as the clinical characteristics, namely, age, body mass index (BMI), estrogen and progesterone receptor expression, and survival.
The samples were collected in accordance with the Declaration of Helsinki and the guidelines issued by the Ethical Committee of the National Cancer Institute of Ukraine. This study was approved by the Ethical www.nature.com/scientificreports/ Committee of RE Kavetsky IEPOR of NASU. Written informed consent was obtained from the living patients, and all protocols were performed following the ethical regulations. Treatment and follow-up data for each patient have been collected from hospital records. All patients were retrospectively followed up from the time of diagnosis.
Histological evaluation. Fresh biopsies were fixed immediately in a neutral buffered 4% formaldehyde solution. Each sample was evaluated using conventional hematoxylin-eosin staining, as described elsewhere. EC samples were graded based on morphological features, according to the 2018 FIGO criteria 31 (FIGO grades 1-3). FIGO stage (FIGO stages I-IV) was also assessed. All tissues were examined by two experienced gynecologic pathologists independently. The presence of metastases was also evaluated.
Immunohistochemistry. The paraffin-embedded tissues were cut (5 µm thick sections) and heated for 15 min at 55 °C prior to paraffin removal in xylene. Three sequential washes with ethanol (99%, 70%, and 30%) removed xylene from the samples. After treatment of tissue sections in a 2% solution of H 2 O 2 in methanol at room temperature for 30 min, they were re-hydrated, and antigens were exposed in a hot citrate buffer (92 °C for 15 min). Samples were stained with the primary rabbit anti-STK4 antibody (HPA015270, Sigma-Aldrich Sweden AB, Stockholm, Sweden) at a dilution of 1:100 in blocking buffer (2% bovine serum albumin, 0.2% Tween-20, 10% glycerol, and 0.05% NaN 3 in phosphate-buffered saline). Next, the immunofluorescence staining was run with Crystal violet; DAPI counterstaining, after the secondary swine anti-rabbit FITC-conjugated antibody (DAKO, Glostrup, Denmark) was applied. Imaging and an image  (16). Note that the highly expressed STK4 in Burkitt lymphoma has only 2 copies (20 × 2 = 2), while in solid tumors there is a gain in copy number values (up to 3 copies in EC cell lines: 20.75 × 2 = 3.3); (D) methylation of the STK4 promoter, obtained by reduced representation bisulfite sequencing (RRBS). The STK4 promoter methylation is quite high in EC, prostate cancer, and breast cancer cell lines, i.e., in hormone-driven cancers of the human reproductive system. RMA robust multichip averaging, relative units of the signal intensity (see detailed description at https:// porta ls. broad insti tute. org/ ccle). AML acute myeloid leukemia, T-cell ALL acute T-cell lymphocytic leukemia, NSC lung non-small cell lung tumor. www.nature.com/scientificreports/ analysis were performed, as described earlier 32 . Staining was evaluated manually, counting the PKN1-positive EC cells. The minimum number of tumor cells we found in any EC tissue sample was 900. Expression was counted in relative units, depending on the intensity of staining.
Statistical analysis. GraphPad Prism software (version 8, GraphPad Software, La Jolla, CA, USA) was used for multiple comparisons of nonparametric criteria. The means of STK4 expression in specimens, obtained by immunohistochemistry were analyzed; negative expression was attributed as 0, low expression (+) as 1, medium levels (++) as 3, and strong signals (+++) as 5. According to tumor grade and FIGO stage, further analyses were performed on the combined mean of each set of ECs. A detailed description of the calculations is given in the figure legends. Briefly, the Wilcoxon matched-pairs test was used to calculate the significance of differences in STK4 expression within and between groups. The Kruskal-Wallis's test for non-parametric values was performed to correlate STK4 expression with tumor grade and FIGO stage. Also, we analyzed the relationship between FIGO stage and patient age, BMI, and the expression of estrogen and progesterone receptors, using the Kruskal-Wallis's test for non-parametric values.
To evaluate a putative dependence between STK4 expression and survival of patients with EC, only EC-specific survival, i.e., only death due to EC, was assessed. The p-value threshold was set below 0.05 in all calculations.
We used the publicly available database Oncomine, which contains published data that have been collected, standardized, annotated, and analyzed by Compendia Bioscience (www. oncom ine. com, March 2021, Thermo Fisher Scientific, Ann-Arbor, MI, USA). Data on STK4 expression was also retrieved from the GTEX Portal (http:// www. gtexp ortal. org/ home) and the Cancer cell line encyclopedia (CCLE) at the Broad Institute website (https:// porta ls. broad insti tute. org/ ccle) on June 20, 2020. An analysis of mutations and expression in cancers was performed using the COSMIC (Catalogue of somatic mutations in cancer) database 33 . Data from the abovementioned databases was not directly correlated with own data on protein expression, but rather served to provide more comprehensive picture on different levels.
The patient's survival rate analysis was performed using the data from the Human Protein Atlas 34 .